Diffraction-based diagnostic devices

ABSTRACT

A biosensor includes a substrate with a layer of receptive material disposed thereon. The receptive material is specific for an analyte of interest. A pattern of active and inactive areas of the receptive material are defined in the receptive material layer by a masking process.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of detectinganalytes in a medium, and more particularly to a process for preparinganalyte-specific diffraction based diagnostic sensors to indicate thepresence of the analyte in a medium.

BACKGROUND

[0002] There are many systems and devices available for detecting a widevariety of analytes in various media. Many of the prior systems anddevices are, however, relatively expensive and require a trainedtechnician to perform the test. A need has been recognized in the artfor biosensor systems that are easy and inexpensive to manufacture, andcapable of reliable and sensitive detection of analytes. Reference ismade, for example, to U.S. Pat. Nos. 5,922,550; 6,060,256; and 6,221,579B1.

[0003] Various advances have been made in the industry for producingbiosensors. For example, U.S. Pat. No. 5,512,131 to Kumar, et al.,describes a device that includes a polymer substrate having a metalcoating. An analyte specific receptor layer is stamped onto the coatedsubstrate. A diffraction pattern is generated when an analyte binds tothe device. A visualization device, such as a spectrometer, is then usedto determine the presence of the diffraction pattern. A drawback to thistype of device is, however, the fact that the diffraction pattern is notdiscernible by the naked eye and, thus, a complex visualization deviceis needed to view the diffraction pattern. Also, the device is generallynot able to detect smaller analytes that do not produce a noticeablediffraction pattern.

[0004] U.S. Pat. No. 5,482,830 to Bogart, et al., describes a devicethat includes a substrate which has an optically active surfaceexhibiting a first color in response to light impinging thereon. Thisfirst color is defined as a spectral distribution of the emanatinglight. The substrate also exhibits a second color which is differentfrom the first color. The second color is exhibited in response to thesame light when the analyte is present on the surface. The change fromone color to another can be measured either by use of an instrument, orby the naked eye. A drawback with the device is, however, the relativelyhigh cost of the device and problems associated with controlling thevarious layers that are placed on the wafer substrate.

[0005] Contact printing techniques have been explored for producingbiosensors having a self-assembling monolayer. U.S. Pat. No. 5,922,550describes a biosensor having a metalized film upon which is printed(contact printed) a specific predetermined pattern of ananalyte-specific receptor. The receptor materials are bound to theself-assembling monolayer and are specific for a particular analyte orclass of analytes. Attachment of a target analyte that is capable ofscattering light to select areas of the metalized plastic film uponwhich the receptor is printed causes diffraction of transmitted and/orreflected light. A diffraction image is produced that can be easily seenwith the eye or, optionally, with a sensing device. U.S. Pat. No.6,060,256 describes a similar device having a metalized film upon whichis printed a specific predetermined pattern of analyte-specificreceptor. The '256 patent is not limited to self-assembling monolayers,but teaches that any receptor which can be chemically coupled to asurface can be used. The invention of the '256 patent uses methods ofcontact printing of patterned monolayers utilizing derivatives ofbinders for microorganisms. One example of such a derivative is a thiol.The desired binding agent can be thiolated antibodies or antibodyfragments, proteins, nucleic acids, sugars, carbohydrates, or any otherfunctionality capable of binding an analyte. The derivatives arechemically bonded to metal surfaces such as metalized polymer films, forexample via a thiol.

[0006] A potential issue of the contact printing techniques describedabove for producing diffraction-based biosensors is the possibility ofcontamination from the print surface (i.e., stamp) during the printingprocess. Also, there is the possibility of uneven application or inkingof the substances due to pressure and contact variations inherent in theprocess, as well as surface energy variations.

[0007] The present invention relates to a biosensor system that is easyand inexpensive to manufacture, is capable of reliable and sensitivedetection of analytes, and avoids possible drawbacks of conventionalmicrocontact printing techniques.

SUMMARY OF THE INVENTION

[0008] Objects and advantages of the invention will be set forth in partin the following description, or may be obvious from the description, ormay be learned through practice of the invention.

[0009] The present invention provides a relatively inexpensive yetsensitive biosensor device, a method for producing such biosensordevices, and a method for detecting analytes of interest present in amedium.

[0010] The biosensor includes a substrate member upon which a layercontaining a receptive material (i.e., biomolecules) has been appliedgenerally uniformly over an entire surface of the substrate member. Thesubstrate may be any one of a wide variety of suitable materials,including plastics, metal coated plastics and glass, functionalizedplastics and glass, silicon wafers, foils, glass, etc. Desirably, thesubstrate is flexible, such as a polymeric film, in order to facilitatethe manufacturing process. The receptive material layer may be appliedby any number of known techniques, including dipping, spraying, rolling,spin coating and any other technique wherein the receptive materiallayer can be applied generally uniformly over the entire test surface ofthe substrate. The invention also includes contact printing methods ofapplying the coating, as long as such methods are conducted in a mannerto prevent inconsistent inking and contamination from contact during theinitial coating process.

[0011] The receptive material layer is defined into a pattern of activeand inactive areas of receptive material in a masking process. Themasking process is based generally on the principle that the receptivematerial biomolecules depend on shape and conformational flexibility forfunction. If the shape or flexibility characteristics are alteredsubstantially, the biomolecules lose their ability to interact or bindwith ligands or analytes having a particular affinity for the molecules(i.e., an antibody-antigen interaction). This principle can be exploitedto form a pattern of active and inactive biomolecules in the receptivematerial layer by a masking process wherein a crosslinking agent isactivated to disrupt or alter the shape and/or flexibilitycharacteristics of the biomolecules in areas exposed by the mask therebyrendering the biomolecules in such areas incapable of interacting withan analyte of interest.

[0012] A generally uniform layer or coating of the cross-linking agentis applied over the receptive material layer. A mask having any desiredpattern of shielded areas and exposed areas (blank, transparent ortranslucent areas) is then placed over the substrate member. The maskand substrate combination are then exposed to a particular stimulus(e.g., light) that activates the crosslinking agent under the exposedareas of the mask. The activated crosslinking agent attaches to thereceptive material, which renders the receptive material inactive in theexposed areas. After the mask is removed, the exposed areas thus definea pattern of inactive areas of the receptive material. The residualunreacted crosslinking agent in the areas that were under the shieldedregions of the mask is removed (e.g., in a washing or rinsing step).

[0013] It should be appreciated that the invention is not limited to anyparticular pattern defined by the mask. Virtually any number andcombination of exposed (active) shapes are possible. In one particularembodiment, the active area pattern is defined by about 10 microndiameter pixels at a spacing of about 5 microns apart over the testsurface of the substrate.

[0014] Upon subsequent exposure of the biosensor to a medium containingan analyte of interest, the analyte binds to the receptive material inthe active areas. The biosensor will then diffract transmitted light ina diffraction pattern corresponding to the active areas. The diffractionpattern may be visible to the naked eye or, optionally, viewed with asensing device. In the case where an analyte does not scatter visiblelight because the analyte is too small or does not have an appreciablerefractive index difference compared to the surrounding medium, adiffraction-enhancing element, such as polymer microparticles, may beused. These micorparticles are coated with a binder or receptivematerial that also specifically binds to the analyte. Upon subsequentcoupling of the analyte to both the patterned biomolecules in thereceptive material layer as well as the microparticles, a diffractionimage is produced which can be easily seen with the eye or, optionally,with a sensing device.

[0015] By “diffraction” it is meant the phenomenon, observed when wavesare obstructed by obstacles, of the disturbance spreading beyond thelimits of the geometrical shadow of the object. The effect is markedwhen the size of the object is of the same order as the wavelength ofthe waves. In the present invention, the obstacles are analytes (with orwithout or attached microparticles) and the waves are light waves.

[0016] In another embodiment of the present invention, nutrients for aspecific class of microorganisms can be incorporated into the receptivematerial layer. In this way, very low concentrations of microorganismscan be detected by first contacting the biosensor of the presentinvention with the nutrients incorporated therein and then incubatingthe biosensor under conditions appropriate for the growth of the boundmicroorganism. The microorganism is allowed to grow until there areenough organisms to form a diffraction pattern.

[0017] The present invention provides a low-cost, disposable biosensorwhich can be mass produced. The biosensors of the present invention canbe produced as a single test for detecting an analyte or it can beformatted as a multiple test device. The uses for the biosensors of thepresent invention include, but are not limited to, detection of chemicalor biological contamination in garments, such as diapers, the detectionof contamination by microorganisms in prepacked foods such as meats,fruit juices or other beverages, and the use of the biosensors of thepresent invention in health diagnostic applications such as diagnostickits for the detection of antigens, microorganisms, and bloodconstituents. It should be appreciated that the present invention is notlimited to any particular use or application.

[0018] These and other features and advantages of the present inventionwill become apparent after a review of the following detaileddescription of the disclosed embodiments.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 is a schematic representation of a method for producingbiosensors according to the invention in a masking process.

DETAILED DESCRIPTION

[0020] The invention will now be described in detail with reference toparticular embodiments thereof. The embodiments are provided by way ofexplanation of the invention, and not meant as a limitation of theinvention. For example, features described or illustrated as part of oneembodiment may be used with another embodiment to yield still a furtherembodiment. It is intended that the present invention include these andother modifications and variations as come within the scope and spiritof the invention.

[0021] The present invention features improved biosensing devices, andmethods for using such biosensing devices, for detecting and quantifyingthe presence or amount of an analyte of interest within a medium. Theanalytes that can be detected by the present invention include, but arenot limited to, microorganisms such as bacteria, yeasts, fungi andviruses. The biosensing devices according to the invention arerelatively inexpensive and have advantages over conventionalmicro-printed biosensors.

[0022] The present invention comprises, in broad terms, a process ofdefining an active pattern of analyte-specific receptor material on asubstrate surface by photo-masking the substrate. A generally uniformcoating or layer of the receptive material is applied to the substratesurface. A generally uniform coating or layer of a crosslinking agent isthen applied over the receptive material layer. A mask is placed overthe substrate, and the mask and substrate combination is exposed to astimulus, such as a light or energy source. In its basic form, the“mask” serves to shield or “protect” at least one area of the receptivematerial from the stimulus or energy source and to expose at least oneadjacent area to the stimulus or energy source so as to activate thecrosslinking agent in the exposed area. For example, the mask may be agenerally transparent or translucent blank (e.g., a strip of material)having any pattern of shielded regions printed or otherwise definedthereon. The exposed or unshielded regions of the mask correspond to theinactive areas of the receptive material layer. Alternatively, the maskmay simply be a single object placed upon the substrate. The area underthe object would be protected and thus define an active area of thereceptive material and the area around the object would be exposed tothe energy source and thus define an area of inactive receptivematerial. The object may alternately have any pattern of openingsdefined therethrough corresponding to the exposed areas.

[0023] The crosslinking agent is “activated” in the exposed areas to theextent that the agent attaches to the biomolecules of the receptivematerial and changes the shape and/or flexibility of the biomoleculereceptor sites to the extent that the biomolecules are thereafterprevented from binding with the analyte of interest in a test medium.The un-activated crosslinking agent in the active receptive materialareas is removed or disassociated from the receptive material by anysuitable process, for example by washing or rinsing the substrate memberwith a solution sufficient to remove the crosslinking agent. Thus, uponremoval of the mask and disassociation of the un-activated crosslinkingagent, a pattern of active and inactive receptive material areas aredefined. It should be understood that “pattern” includes as few as oneactive area and one inactive area.

[0024] Upon subsequent exposure of the biosensor to a medium containingthe analyte of interest, such analyte will bind to the receptors in theactive receptive material areas. The analyte facilitates diffraction oftransmitted and/or reflected light in a visible diffraction patterncorresponding to the active areas. As discussed in greater detail below,an enhancer may be used for enhancing diffraction from extremely smallanalytes.

[0025] The crosslinking agent may be any one of a number of commerciallyavailable homobifunctional agents wherein both ends of the agent arephotosensitive, one example of which is(bis-[β-(4-azidosalicylamido)ethyl]disulfide). Such crosslinking agentsare typically available in powder form and, in order to coat thereceptive material layer, it may be necessary to put the agent insolution with water or a buffer. The solution is applied over thereceptive material by any appropriate method, such as dipping, spraying,coating, etc. The solution is then dried leaving a relatively thin drycoat of the agent over the receptive material. It should be ensured thatthe concentration of the agent is sufficient to essentially cover thereceptive material. This is particularly important because thecrosslinking agent is non-specific with respect to the receptivematerial. In other words, there is no particular attraction or affinitybetween the crosslinking agent and the receptive material.Alternatively, it may be desired to maintain the crosslinking agent insolvent form during the masking process. For example, a small amount ofsolution containing an adequate concentration of the crosslinking agentmay be “sandwiched” between the substrate and a clear film or slideprior to placing the mask against the substrate.

[0026] Upon being exposed to a light source through the exposed regionsof the mask, the ends of the cross-linking agent affix to thebiomolecules in the receptive material layer and block or distort thereceptor sites to the extent that the biomolecules are renderedincapable of subsequently interacting with the analyte of interest.

[0027] It should be appreciated that, due to the photosensitive natureof the crosslinking agent, it may be desired to conduct the processes ofapplying and masking the agent are done in a light protectedenvironment.

[0028] The mask is then removed from the substrate member, desirably ina light protected environment, and the unactivated crosslinking agent isdisassociated from the receptive material in the active areas. This maybe done in various ways, including rinsing the substrate with a bufferor water.

[0029] The analytes that are contemplated as being detected using thepresent invention include, but are not limited to, bacteria; yeasts;fungi; viruses; rheumatoid factor; antibodies, including, but notlimited to IgG, IgM, IgA, IgD, and IgE antibodies; carcinoembryonicantigen; streptococcus Group A antigen; viral antigens; antigensassociated with autoimmune disease, allergens; tumor antigens;streptococcus Group B antigen; HIV I or HIV II antigen; or host response(antibodies) to these and other viruses; antigens specific to RSV orhost response (antibodies) to the virus; antigen; enzyme; hormone;polysaccharide; protein; lipid; carbohydrate; drug or nucleic acid;Salmonella species; Candida species, including, but not limited toCandida albicans and Candida tropicalis; Neisseria meningitides groupsA, B, C, Y and W sub 135; Streptococcus pneumoniae; E. coli; Haemophilusinfluenza type A/B; an antigen derived from microorganisms; prostratespecific antigen (PSA) and C-reactive protein (CRP) antigen; a hapten; adrug of abuse; a therapeutic drug; an environmental agent; and antigensspecific to Hepatitis. In broad terms, the “analyte of interest” may bethought of as any agent whose presence or absence from a biologicalsample is indicative of a particular health state or condition.

[0030] It is also contemplated that nutrients for a specific class ofmicroorganism can be incorporated into the receptive material layer. Inthis way, very low concentrations of microorganisms can be detected byexposing the biosensor of the present invention with the nutrientsincorporated therein to the suspect medium and then incubating thebiosensor under conditions appropriate for the growth of the boundmicroorganism. The microorganisms are allowed to grow until there areenough organisms to form a diffraction pattern. Of course, in somecases, the microorganism is present or can multiply enough to form adiffraction pattern without the presence of a nutrient in the activereceptive material areas.

[0031] The receptive material is characterized by an ability tospecifically bind the analyte or analytes of interest. The variety ofmaterials that can be used as receptive material is limited only by thetypes of material which will combine selectively (with respect to anychosen sample) with a secondary partner. Subclasses of materials whichfall in the overall class of receptive materials include toxins,antibodies, antibody fragments, antigens, hormone receptors, parasites,cells, haptens, metabolites, allergens, nucleic acids, nuclearmaterials, autoantibodies, blood proteins, cellular debris, enzymes,tissue proteins, enzyme substrates, coenzymes, neuron transmitters,viruses, viral particles, microorganisms, proteins, polysaccharides,chelators, drugs, aptamers, peptides, and any other member of a specificbinding pair. This list only incorporates some of the many differentmaterials that can be coated onto the substrate surface to produce athin film assay system. Whatever the selected analyte of interest is,the receptive material is designed to bind specifically with the analyteof interest.

[0032] The matrix or medium containing the analyte of interest may be aliquid, a solid, or a gas, and can include a bodily fluid such asmucous, saliva, urine, fecal material, tissue, marrow, cerebral spinalfluid, serum, plasma, whole blood, sputum, buffered solutions, extractedsolutions, semen, vaginal secretions, pericardial, gastric, peritoneal,pleural, or other washes and the like. The analyte of interest may be anantigen, an antibody, an enzyme, a DNA fragment, an intact gene, a RNAfragment, a small molecule, a metal, a toxin, an environmental agent, anucleic acid, a cytoplasm component, pili or flagella component,protein, polysaccharide, drug, or any other material. For example,receptive material for bacteria may specifically bind a surface membranecomponent, protein or lipid, a polysaccharide, a nucleic acid, or anenzyme. The analyte which is specific to the bacteria may be apolysaccharide, an enzyme, a nucleic acid, a membrane component, or anantibody produced by the host in response to the bacteria. The presenceor absence of the analyte may indicate an infectious disease (bacterialor viral), cancer or other metabolic disorder or condition. The presenceor absence of the analyte may be an indication of food poisoning orother toxic exposure. The analyte may indicate drug abuse or may monitorlevels of therapeutic agents.

[0033] One of the most commonly encountered assay protocols for whichthis technology can be utilized is an immunoassay. However, the generalconsiderations apply to nucleic acid probes, enzyme/substrate, and otherligand/receptor assay formats. For immunoassays, an antibody may serveas the receptive material or it may be the analyte of interest. Thereceptive material, for example an antibody or an antigen, should form astable, reactive layer on the substrate surface of the test device. Ifan antigen is to be detected and an antibody is the receptive material,the antibody must be specific to the antigen of interest; and theantibody (receptive material) must bind the antigen (analyte) withsufficient avidity that the antigen is retained at the test surface. Insome cases, the analyte may not simply bind the receptive material, butmay cause a detectable modification of the receptive material to occur.This interaction could cause an increase in mass at the test surface ora decrease in the amount of receptive material on the test surface. Anexample of the latter is the interaction of a degradative enzyme ormaterial with a specific, immobilized substrate. In this case, one wouldsee a diffraction pattern before interaction with the analyte ofinterest, but the diffraction pattern would disappear if the analytewere present. The specific mechanism through which binding,hybridization, or interaction of the analyte with the receptive materialoccurs is not important to this invention, but may impact the reactionconditions used in the final assay protocol.

[0034] In addition to producing a simple diffraction image, patterns ofanalytes can be such as to allow for the development of a holographicsensing image and/or a change in visible color. Thus, the appearance ofa hologram or a change in an existing hologram will indicate a positiveresponse. The pattern made by the diffraction of the transmitted lightcan be any shape including, but not limited to, the transformation of apattern from one pattern to another upon binding of the analyte to thereceptive material. In particularly preferred embodiments, thediffraction pattern becomes discernible in less than one hour aftercontact of the analyte with the biosensing device of the presentinvention.

[0035] The diffraction grating which produces the diffraction of lightupon interaction with the analyte must have a minimum periodicity ofabout ½ the wavelength and a refractive index different from that of thesurrounding medium. Very small analytes, such as viruses or molecules,can be detected indirectly by using a larger, “diffraction-enhancingelement,” such as a microparticle, that is specific for the smallanalyte. One embodiment in which the small analyte can be detectedcomprises coating the enhancing particle, such as a latex bead orpolystyrene bead, with a receptive material, such as an antibody, thatspecifically binds to the analyte of interest. Particles that can beused in the present invention include, but are not limited to, glass,cellulose, synthetic polymers or plastics, latex, polystyrene,polycarbonate, proteins, bacterial or fungal cells, silica, celluloseacetate, carbon, and the like. The particles are desirably spherical inshape, but the structural and spatial configuration of the particles isnot critical to the present invention. For instance, the particles couldbe slivers, ellipsoids, cubes, random shape and the like. A desirableparticle size ranges from a diameter of approximately 0.1 micron to 50microns, desirably between approximately 0.1 micron and 2.0 microns. Thecomposition of the particle is not critical to the present invention.

[0036] Desirably, the receptive material layer on the substrate willspecifically bind to an epitope on the analyte that is different fromthe epitope used in the binding to the enhancing particle. Thus, fordetecting a small analyte, such as viral particles, in a medium, themedium is first exposed to the latex particles having the virus-specificreceptive material thereon. The small analytes of interest in the mediumwill bind to the latex particles. Then, the latex particles areoptionally washed and exposed to the biosensor film with the pattern ofactive receptive material areas containing the virus-specificantibodies. The antibodies then bind to the viral particles on the latexbead thereby immobilizing the latex beads in the same pattern as theactive areas on the film. Because the bound latex beads will causediffraction of the visible light, a diffraction pattern is formed,indicating the presence of the viral particle in the liquid. Othercombinations using diffraction enhancing particles are described, forexample, in U.S. Pat. No. 6,221,579 incorporated herein for allpurposes.

[0037] Any one of a wide variety of materials may serve as the substrateto which the receptive material is applied. Such materials are wellknown to those skilled in the art. For example, the substrate may beformed of any one of a number of suitable plastics, metal coatedplastics and glass, functionalized plastics and glass, silicon wafers,foils, glass, etc. Rather than requiring a rigid substrate for thephotopatterning process described herein, it has been found thatthermoplastic films are quite suitable. Such films include, but are notlimited to, polymers such as: polyethylene-terephthalate (MYLAR®),acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, cellulose triacetate, polyethylene,polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, and aromatic polysulfones. Preferably, the plasticfilm has an optical transparency of greater than 80 percent. Othersuitable thermoplastics and suppliers may be found, for example, inreference works such as the Modern Plastics Encyclopedia (McGraw-HillPublishing Co., New York 1923-1996).

[0038] In one embodiment of the invention, the thermoplastic film mayhave a metal coating. The film with metal coating thereon may have anoptical transparency of between approximately 5 percent and 95 percent.A more desired optical transparency for the thermoplastic film used inthe present invention is between approximately 20 percent and 80percent. In a desired embodiment of the present invention, thethermoplastic film has at least an approximately 80 percent opticaltransparency, and the thickness of the metal coating is such as tomaintain an optical transparency greater than about 20 percent, so thatdiffraction patterns can be produced by either reflected or transmittedlight. This corresponds to a metal coating thickness of about 20nanometers. However, in other embodiments of the invention, the metalthickness may be between approximately 1 nanometer and 1000 nanometers.

[0039] The preferred metal for deposition on the film is gold. However,silver, aluminum, chromium, copper, iron, zirconium, platinum, titanium,and nickel, as well as oxides of these metals, may be used. Chromiumoxide can be used to make metalized layers.

[0040] The receptive material and crosslinking agent may be applied tothe substrate by any conventional method. The material is applied sothat it generally uniformly covers an entire (for example, upper)surface of the substrate. Non-contact methods for applying the receptivematerial may be desired so as to eliminate the possibility ofcontamination by contact during application. Suitable applicationmethods include, but are not limited to, dipping, spraying, rolling,spin coating, and any other technique wherein the receptive materiallayer and blocking agent can be applied generally uniformly over theentire test surface of the substrate. Simple physisorption can occur onmany materials, such as polystryene, glass, nylon, or other materialswell known to those skilled in the art. One particular embodiment ofimmobilizing the analyte-specific receptive material layer involvesmolecular attachment, such as that possible between thiol ordisulfide-containing compounds and gold. Typically, a gold coating ofabout 5 to about 2000 nanometers thick is supported on a silicon wafer,glass, or polymer film (such as a MYLAR® film). The analyte-specificreceptor attaches to the gold surface upon exposure or spraying of asolution of the receptive material.

[0041] Although not preferred, the invention also includes contactprinting methods of applying the receptive material and crosslinkingagent.

[0042] The technique selected should minimize the amount of receptivematerial required for coating a large number of test surfaces andmaintain the stability/functionality of the receptive material duringapplication. The technique should also apply or adhere the receptivematerial to the substrate in a uniform and reproducible fashion.

[0043] It is also contemplated that the receptive material layer may beformed on the substrate as a self-assembling monolayer ofalkanethiolates, carboxylic acids, hydroxamic acids, and phosphonicacids on metalized thermoplastic films. The self-assembling monolayershave receptive material bound thereto. Reference is made to U.S. Pat.No. 5,922,550 for a more detailed description of such self-assemblingmonolayers and methods for producing the monolayers. The '550 patent isincorporated herein in its entirety for all purposes.

[0044] The mask may be formed of any suitable material that protects orblocks portions of the underlying substrate from the irradiatingstimulus or energy source. A material that has proven useful fordefining patterns of active and inactive receptive material regions on agold-plated MYLAR® film coated with an antibody solution is atransparent or translucent polymer film (such as MYLAR®) having apattern of shielded or protected regions printed thereon. This type ofmask is useful for light sources with a wavelength equal to or greaterthan about 330 nanometers. For light sources having a wavelength belowabout 330 nanometers, a quartz or fused silica mask having chrome platedshielded regions defined thereon may be used. The mask may define anypattern of active and inactive regions of the receptive material. Theactive regions creating the visible diffraction pattern may be ofvirtually any size, shape, and pattern. It may be desired to select asize and pattern so as to maximize the visible diffraction contrastbetween the active and inactive regions. As one example, it has beenfound suitable if the active regions are defined as generally circularwith a diameter of about 10 microns and spaced from each other by about5 microns.

[0045]FIG. 1 is a schematic representation of one method for producingbiosensors according to the invention. Step A represents a receptivematerial layer 2 applied to a substrate member 4. Step B represents acrosslinking agent 6 as described above applied to the substrate member4 such that the crosslinking agent 6 generally uniformly covers thereceptive material 2. Step C represents a mask 8 disposed over thesubstrate member 4. The mask 8 includes exposed or open regions 12 andshielded or protected regions 10 defined thereon. Step D represents themask 8 and substrate member 4 combination being irradiated with a lightsource 14. It can be seen that the areas of the substrate member 4underlying the shielded regions 10 of the mask 8 are protected from thelight source 14. The crosslinking agent 6 exposed to the light source 14through the open regions 12 of the mask 8 is activated by the energysource 14 so that the ends of the cross-linking agent 6 affix to theindividual biomolecules of the receptive material 2. Activated orcrosslinked areas 16 are defined in a pattern corresponding to thepattern of the exposed regions 12 of the mask 8, and unactivated oruncrosslinked areas 18 are defined in a pattern corresponding to thepattern of the shielded regions 10 of the mask 8. Step E represents thebiosensor after the mask 8 is removed and prior to the step of removingor disassociating the unactivated crosslinking agent 6 from the regions18. Step F represents the biosensor in its final form after theunactivated crosslinking agent 6 has been removed (for example, bywashing). The biosensor includes active areas 20 of the receptivematerial and inactive areas 22.

[0046] The biosensors according to the invention have a wide range ofuses in any number of fields. The uses for the biosensors of the presentinvention include, but are not limited to, detection of chemical orbiological contamination in garments, such as diapers, generally thedetection of contamination by microorganisms in prepacked foods such asmeats, fruit juices or other beverages, and the use of the biosensors ofthe present invention in health diagnostic applications such asdiagnostic kits for the detection of proteins, hormones, antigens,nucleic acids, DNA, microorganisms, and blood constituents. The presentinvention can also be used on contact lenses, eyeglasses, window panes,pharmaceutical vials, solvent containers, water bottles, band-aids,wipes, and the like to detect contamination. In one embodiment, thepresent invention is contemplated in a dipstick form in which thepatterned substrate is mounted at the end of the dipstick. In use thedipstick is dipped into the liquid in which the suspected analyte may bepresent and allowed to remain for several minutes. The dipstick is thenremoved and then, either a light is projected through the substrate orthe substrate is observed with a light reflected from the substrate. Ifa diffraction pattern is observed, then the analyte is present in theliquid.

[0047] In another embodiment of the present invention, a multipleanalyte test is constructed on the same support. A strip may be providedwith several patterned substrate sections. Each section has a differentreceptive material that is different for different analytes. It can beseen that the present invention can be formatted in any array with avariety of patterned substrates thereby allowing the user of thebiosensor device of the present invention to detect the presence ofmultiple analytes in a medium using a single test.

[0048] In yet another embodiment of the present invention, the biosensorcan be attached to an adhesively backed sticker or decal which can thenbe placed on a hard surface or container wall. The biosensor can beplaced on the inside surface of a container such as a food package or aglass vial. The biosensor can then be visualized to determine whetherthere is microbial contamination.

[0049] It should be understood that resort may be had to various otherembodiments, modifications, and equivalents of the embodiments of theinvention described herein, which, after reading this description of theinvention, may suggest themselves to those skilled in the art withoutdeparting from the scope and spirit of the present invention.

What is claimed is:
 1. A biosensor, comprising: a substrate member; areceptive material layer generally uniformly covering a side of saidsubstrate member, said receptive material being specific for an analyteof interest; a pattern of at least one active area and at least oneinactive area of said receptive material defined in said receptivematerial layer, said receptive material in said inactive area renderedinactive by a crosslinking agent; wherein said active and inactive areasare defined in a masking process wherein the crosslinking agent isapplied generally uniformly over said receptive material layer and amask is then placed over said substrate member prior to exposing saidsubstrate member to a stimulus for activating said crosslinking agent,areas underlying shielded regions of the mask defining said active areasand areas exposed by the mask having the crosslinking agent activated bythe stimulus and defining said inactive areas; and wherein when saidbiosensor is exposed to a medium containing said analyte of interest,the analyte binds to said receptive material in said active areas andfacilitates subsequent diffraction of transmitted or reflected light ina diffraction pattern corresponding to said active areas.
 2. Thebiosensor as in claim 1, wherein said pattern of active and inactiveareas comprises a plurality of said active areas and a plurality of saidinactive areas defined by said masking process.
 3. The biosensor as inclaim 1, wherein said crosslinking agent in said active areas is removedin a washing process.
 4. The biosensor as in claim 1, wherein saidcrosslinking comprises a homobifunctional agent in which both ends ofsaid agent are photosensitive, said stimulus being a light source at awavelength sufficient to activate said ends of said cross-linking agent.5. The biosensor as in claim 4, wherein said crosslinking agentcomprises (bis-[β-(4-azidosalicylamido)ethyl]disulfide).
 6. Thebiosensor as in claim 1, wherein said substrate comprises a materialfrom the list of materials consisting of plastics, metal coated plasticsand glass, functionalized plastics and glass, silicon wafers, glass, andfoils.
 7. The biosensor as in claim 1, wherein said substrate membercomprises a polymer film coated with a metal.
 8. The biosensor as inclaim 7, wherein said polymer film comprises polyethylene-terephthalate.9. The biosensor as in claim 7, wherein said metal is selected from thegroup consisting of gold, silver, chromium, nickel, platinum, aluminum,iron, copper, gold oxide, titanium, titanium oxide, chromium oxide orzirconium.
 10. The biosensor as in claim 7, wherein said metal is gold.11. The biosensor as in claim 1, wherein said diffraction pattern isvisible to the naked eye.
 12. The biosensor as in claim 1, wherein saidreceptive material is at least one of antigens, antibodies, nucleotides,chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili,bacterial flagellar materials, nucleic acids, polysaccharides, lipids,proteins, carbohydrates, metals, hormones, aptamers, peptides, andrespective receptors for said materials.
 13. The biosensor as in claim1, wherein said analyte of interest is at least one of a bacteria,yeast, fungus, virus, rheumatoid factor, IgG, IgM, IgA, IgD, and IgEantibodies, carcinoembryonic antigen, streptococcus Group A antigen,viral antigens, antigens associated with autoimmune disease, allergens,tumor antigens, streptococcus group B antigen, HIV I or HIV II antigen,antib ies viruses, antigens specific to RSV, an antibody, antigen,enzyme, hormone, polysaccharide, protein, lipid, carbohydrate, drug,nucleic acid, Neisseria meningitides groups A, B, C, Y and W sub 135,Streptococcus pneumoniae, E. coli K1, Haemophilus influenza type A/B, anantigen derived from microorganisms, PZA and CRP antigens, a hapten, adrug of abuse, a therapeutic drug, an environmental agents, or antigensspecific to Hepatitis.
 14. A method of making a biosensor, comprisingthe steps of: forming a receptive material layer generally uniformlyover a surface of a substrate member, the layer containing a receptivematerial specific for an analyte of interest; applying an activatablecrosslinking agent generally uniformly over the receptive materiallayer; placing a mask over the substrate member, the mask having aconfiguration so as to cover at least one underlying area of thesubstrate while exposing at least one adjacent area; exposing the maskand substrate to a stimulus for activating the crosslinking agent suchthat the crosslinking agent in the exposed areas affixes to anddeactivates the receptive material; removing the mask from the substrateand disassociating the unactivated crosslinking agent from the receptivematerial in the areas that were covered by the mask to define activeareas of the receptive material; wherein a resulting pattern of activeand inactive areas of the receptive material are defined such that whenthe biosensor is exposed to a medium containing the analyte of interest,the analyte binds to the receptive material in the active areas andfacilitates subsequent diffraction of transmitted or reflected light ina diffraction pattern corresponding to the active areas.
 15. The methodas in claim 14, comprising defining a pattern of the active and inactiveareas with the mask, the pattern comprising a plurality of the activeareas and a plurality of the inactive areas.
 16. The method as in claim14, wherein the crosslinking agent is a homobifunctional agent in whichboth ends of said agent are photosensitive.
 17. The method as in claim16, comprising activating the crosslinking agent with a light source.18. The method as in claim 16, wherein the crosslinking agent comprises(bis-[β-(4-azidosalicylamido)ethyl]disulfide).
 19. The method as inclaim 14, comprising disassociating the crosslinking agent in the activeareas by washing the substrate member with a solution sufficient toremove the crosslinking agent from the substrate member.
 20. The methodas in claim 14, comprising selecting the substrate member from the groupof materials consisting of plastics, metal coated plastics and glass,functionalized plastics and glass, silicon wafers, glass, and foils. 21.The method as in claim 14, wherein the substrate member comprises apolymer film coated with a metal.
 22. The method as in claim 21, whereinthe polymer film comprises polyethylene-terephthalate.
 23. The method asin claim 21, comprising selecting the metal from the group consisting ofgold, silver, chromium, nickel, platinum, aluminum, iron, copper,titanium, titanium oxide, gold oxide, chromium oxide and zirconium. 24.The method as in claim 21, wherein the metal is gold.
 25. The method asin claim 14, comprising viewing the diffraction pattern of active areaswith the naked eye.
 26. The method as in claim 14, comprising selectingthe receptive material from at least one of antigens, antibodies,nucleotides, chelators, enzymes, bacteria, yeasts, fungi, viruses,bacterial pili, bacterial flagellar materials, nucleic acids,polysaccharides, lipids, proteins, carbohydrates, metals, hormones,aptamers, peptides, and respective receptors for said materials.
 27. Themethod as in claim 14, wherein the analyte of interest is selected fromat least one of a bacteria, yeast, fungus, virus, rheumatoid factor,IgG, IgM, IgA, IgD, and IgE antibodies, carcinoembryonic antigen,streptococcus Group A antigen, viral antigens, antigens associated withautoimmune disease, allergens, tumor antigens, streptococcus group Bantigen, HIV I or HIV II antigen, antibodies viruses, antigens specificto RSV, an antibody, antigen, enzyme, hormone, polysaccharide, protein,lipid, carbohydrate, drug, nucleic acid, Neisseria meningitides groupsA, B, C, Y and W sub 135, Streptococcus pneumoniae, E. coli K1,Haemophilus influenza type A/B, an antigen derived from microorganisms,PSA and CRP antigens, a hapten, a drug of abuse, a therapeutic drug, anenvironmental agents, or antigens specific to Hepatitis.